CN114306386A - Application of antler stem cell conditioned medium - Google Patents
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Abstract
The invention relates to the technical field of traditional Chinese medicinal material application, in particular to application of a pilose antler stem cell conditioned medium. The research of the invention shows that the antler stem cell conditioned medium can effectively promote periodontal bone defect caused by simulated periodontitis environment, promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells and inhibit the formation of osteoclast. Therefore, the antler stem cell conditioned medium can be prepared into medicines, health products or washing products for treating periodontitis accompanied with periodontal bone defect, and can also be used for treating other bone injuries.
Description
Technical Field
The invention relates to the technical field of traditional Chinese medicinal material application, in particular to application of a pilose antler stem cell conditioned medium.
Background
Periodontitis is a common chronic inflammatory disease that often results in damage to periodontal supporting tissues, including the gingiva, periodontal ligament, and alveolar bone. Severe periodontitis can eventually cause tooth loss due to severe alveolar bone destruction, which seriously affects people's health and quality of life. Because the periodontal tissue lacks self-repairing reconstruction ability, the regeneration of alveolar bone tissue cannot be realized in the true sense by the traditional periodontal treatment mode. Therefore, it is necessary to find an effective method for regenerating periodontal bone tissue in the treatment of periodontitis accompanied with destruction of periodontal bone tissue.
In recent years, with the rapid development of stem cell transplantation technology, a very promising therapeutic method is provided for bone regeneration, but the application of cell transplantation in clinic is limited due to the low cell survival rate and short survival time after transplantation. With the progress of research, it is gradually recognized that many factors produced by mesenchymal stem cells through paracrine action play a major role in cell therapy. Paracrine proteins are secreted by cells and play an important role in intercellular communication, have the advantages of easy collection, easy storage and transportation, controllable quality, easier standardization and the like, and can play a therapeutic role similar to stem cell transplantation when transplanted into an animal model.
At present, the more commonly used stem cells are mesenchymal stem cells, and bone marrow, umbilical cord and adipose tissue are proved to be main tissue sources of the mesenchymal stem cells, but because the mesenchymal stem cells have limited sources and limited passage times during in vitro culture, the wide application in tissue regeneration is seriously limited, so that the finding of the stem cells which can stably provide a large amount of paracrine proteins with therapeutic action is a key problem for realizing important breakthrough in the field of cell-free regeneration reagent development.
Cornu cervi pantotrichum is the only known mammalian organ which can be completely regenerated after falling off and consists of tissues such as cartilage, bones, skin, blood vessels, nerves and the like. The pilose antler as one bone organ has very high growth rate (up to 2cm/d) and can repair and regenerate completely in natural condition. More surprisingly, it was found that pilose antler regeneration is a stem cell-based process. The periodical regeneration of antler from horn handle (permanent bone stake born on the frontal bone of deer head) every year depends entirely on the proliferation and differentiation of horn handle periosteum cells, which have been proved to be mesenchymal stem cells with certain embryonic stem cell properties and are defined as antler stem cells. The unique attribute of the pilose antler stem cell that has the capacity of developing into a complete bone accessory organ is absolutely unique in mammals, and the application of the pilose antler stem cell in the field of bone regeneration is suggested. The early experiments show that compared with the common mesenchymal stem cells, the antler stem cells have stronger proliferation capacity, can be stably passed to 50 generations and keep stable proliferation activity, and the conditioned medium obtained by culturing the antler stem cells has various physiological activities, but no research shows that the antler stem cell conditioned medium has a repairing effect on periodontitis, particularly damage of periodontal support tissues.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide the use of the antler stem cell conditioned medium for the treatment of periodontitis.
The invention provides application of a stem cell conditioned medium under the condition of pilose antler in preparing a medicament for treating bone injury.
In some embodiments, the bone injury comprises a periodontal bone injury.
The invention also provides application of the antler stem cell conditioned medium in preparing products for promoting stem cell proliferation. In some embodiments, the stem cell is a bone marrow mesenchymal stem cell.
The invention also provides application of the antler stem cell conditioned medium in preparing products for promoting osteogenic differentiation of stem cells. In some embodiments, the stem cell is a bone marrow mesenchymal stem cell.
The invention also provides application of the antler stem cell conditioned medium in preparing products for inhibiting differentiation of monocytes into osteoclasts.
In the invention, the preparation method of the antler stem cell conditioned medium comprises the following steps: and (3) centrifugally separating the antler stem cell culture product to obtain a supernatant, and performing ultrafiltration concentration to obtain the antler stem cell conditioned medium.
In some embodiments, the ultrafiltration concentration has a molecular weight cut-off of 3 KDa. The ultrafiltration conditions included centrifugation at 5000g at 4 ℃.
In some embodiments, the ultrafiltration concentration further comprises a step of freeze-drying after filtration to obtain freeze-dried powder, and the pre-cooling temperature of the freeze-drying is-45 ℃. The filtrate was frozen overnight at-80 ℃ before lyophilization. Namely freezing for 8-12 h at-80 ℃.
The invention also provides a product for treating periodontitis, which comprises the antler conditioned stem cell conditioned medium.
In some embodiments, the product for treating periodontitis according to the present invention comprises lyophilized powder of pilose antler-conditioned stem cell-conditioned medium.
In other embodiments, the product for treating periodontitis according to the present invention comprises lyophilized powder of pilose antler conditioned stem cell conditioned medium and PBS buffer. In this example, the mass-to-volume ratio of the lyophilized powder to PBS buffer was 1g: (10-20) mL.
The invention also provides a method for treating periodontitis, which comprises the step of administering the product for treating periodontitis.
The research of the invention shows that the antler stem cell conditioned medium can effectively promote periodontal bone defect caused by simulated periodontitis environment, promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells and inhibit the formation of osteoclast. Therefore, the antler stem cell conditioned medium can be prepared into medicines, health products or washing products for treating periodontitis accompanied with periodontal bone defect, and can also be used for treating other bone injuries.
Drawings
FIG. 1 shows the results of HE staining for treatment of periodontal bone defects in rats;
FIG. 2 shows the statistical results of micro CT detection of periodontal bones of rats;
FIG. 3 shows the result of CCK-8 detecting the proliferation of mesenchymal stem cells;
FIG. 4 shows the results of alkaline phosphatase staining;
FIG. 5 shows alizarin Red staining results;
FIG. 6 shows the results of staining with tartrate-resistant acid phosphatase.
Detailed Description
The invention provides the application of the conditioned medium of the pilose antler stem cells, and the technical personnel in the field can appropriately improve the technological parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention discloses a pilose antler stem cell conditioned medium, which is a medium cultured with pilose antler stem cells and contains substances secreted by all the pilose antler stem cells to the outside of cells.
The research proves that the conditioned medium can effectively promote periodontal bone defect caused by simulated periodontitis environment, so that the conditioned medium can be prepared into medicines, health-care products or washing products for treating periodontitis accompanied with periodontal bone defect and can also be used for treating other bone injuries.
The invention is further illustrated by the following examples:
example 1 preparation of conditioned Medium for deer antler Stem cells
1. Cultivation of pilose antler stem cells
The preparation method of the deer antler stem cells is obtained by separating the Cervus nippon hornstalk periosteum, and the specific preparation method refers to the literature: cell Death and Disease of a mammalian organ-der organ, (2019)10:443, among Datao Wang, Debbie Berg, Hengxing Ba, HongmeiSun, Zhen Wang and ChunyiLi, and De anti stemcell are of a novel type of cell that is a cell Death function regeneration.
Culturing cornu Cervi Pantotrichum stem cells in culture flask at 37 deg.C and 5% CO2The incubators were incubated in complete medium, with the composition containing DMEM basal medium (Gibco), 10% fetal bovine serum (Gibco), 1% penicillin/streptomycin (Thermo Fisher Scientific).
2. Collection and concentration of pilose antler stem cell conditioned medium
2.1 when the antler stem cells grow to more than 90 percent of confluence, PBS (Thermo Fisher Scientific) is cleaned once, pancreatin (Thermo Fisher Scientific) is added for digestion for 2 minutes, complete culture medium is added to stop digestion, the cells are collected into a centrifuge tube, centrifugation is carried out at 1000 Xg, supernatant is removed, the cells are resuspended, the cells are passed to a new culture bottle, complete culture medium is added, the complete culture medium is removed when the cells are cultured to 70 to 80 percent of confluence, PBS is cleaned for 2 to 3 times, the conditioned medium without fetal bovine serum (the components are alpha-MEM culture medium and 1 percent penicillin/streptomycin) is replaced for continuous culture, cell culture supernatant is collected after 48 hours, centrifugation is carried out at 1000 Xg for 5 minutes, and exfoliated cells and fragments are removed, thus obtaining the antler stem cell conditioned medium.
2.2 the conditioned medium obtained was concentrated by ultrafiltration using a 3KD ultrafiltration tube at 5000 Xg at 4 ℃ while the same volume of alpha-MEM medium was used as an unloaded control and concentrated by ultrafiltration using 5000 Xg at 4 ℃ on the same alpha-MEM medium.
2.3 filtering and sterilizing the concentrated antler stem cell conditioned medium and the alpha-MEM medium by using a 0.22 mu m filter membrane, placing the filtered and sterilized antler stem cell conditioned medium and the alpha-MEM medium in a clean and sterile shallow siliconized glass dish, horizontally placing the sterilized antler stem cell conditioned medium and the alpha-MEM medium in an ultra-low temperature refrigerator at minus 80 ℃ after sealing the mouth of the filter membrane, and freezing the sterilized antler stem cell conditioned medium and the alpha-MEM medium overnight.
2.4 freeze-drying: opening a vacuum freeze dryer in advance to pre-cool to-45 deg.C, drying the frozen cornu Cervi Pantotrichum dry cell conditioned medium in the vacuum freeze dryer, pulverizing at low temperature after drying to obtain lyophilized powder containing cornu Cervi Pantotrichum dry cell conditioned medium, sealing, and storing at room temperature in a dry place with humidity not more than 50%.
2.5 redissolution: PBS was added to each of the in vivo and in vitro experiments of examples 2 and 3 at a mass-to-volume ratio of 1g:10mL and 1g:20mL for rehydration before carrying out the in vivo and in vitro experiments (i.e., 1g:10mL for rehydration when the lyophilized powder is used in the following experiments for treating periodontal bone defects in rats in vivo and 1g:20mL for rehydration when the in vitro experiments are carried out), and the mixture was mixed until dissolved to prepare a concentrated solution of a deer antler stem cell conditioned medium (ASC-CM) and an alpha-MEM medium.
Example 2 antler stem cell conditioned Medium promotes regenerative repair of periodontal bone defects
1. Construction of rat periodontal bone defect model for simulating periodontitis environment
30 SD male rats of 6 weeks old are purchased, observed for one week by routine feeding, and subjected to surgical modeling. Intraperitoneal injection of 1% sodium pentobarbital for anesthesia, conventional disinfection with alcohol and iodine, surgical exposure of the buccal bone plate of the first molar of the left lower jaw, removal of the buccal bone plate by a turbo machine (range: horizontally from the mesial root of the first molar to the mesial root of the second molar, vertically from the apical end of the alveolar ridge to the apical end), exposure of the root surface of the first molar, 3mm wide of the defect area, 2mm high and 1mm deep.
2. Post-operative treatment
Before operation, the sterile collagen membrane was cut into 3mm × 2mm, and 50 μ l of PBS, α -MEM, and ASC-CM were added dropwise to infiltrate the membrane. The 30 rats were randomly divided into three groups, the treated collagen membrane was implanted into the defect area, i.e., PBS group, α -MEM group and ASC-CM group, respectively, the mucosa was replaced and sutured to cover the defect, all animals were fed with soft chow, and the prophylactic antibiotics were injected for 3 days. The drug was administered once a week, three groups each time were injected topically at the defect with 50 μ LPBS, α -MEM, ASC-CM, and two times post-surgery.
3. Tissue sampling
And in the 4 th week after the operation, excessive anesthetic is injected to kill the rat, the mandible of the rat is obtained, the cleaned mandible is put into 4 percent paraformaldehyde for fixation, whether the operation area is healed or not is noticed when a specimen is obtained, abnormality exists or not, and the like, HE staining detection and Micro-CT scanning detection are carried out on the repaired tissue, and the treatment effect is evaluated.
HE staining
Fixing the bone tissue sample collected in the above steps in 4% paraformaldehyde for 48 hours, washing with running water for 6 hours, decalcifying for one week by formic acid decalcifying solution, washing with PBS, dehydrating, embedding paraffin, slicing, baking at 63 ℃ for 3 hours, dewaxing with xylene and gradient alcohol, rehydrating, staining with hematoxylin, turning blue, staining with eosin, dehydrating, sealing, and taking pictures by microscope observation.
The result is shown in figure 1, the PBS group defect area has a small amount of new tissues, the alpha-MEM group defect area also has the new tissues to cover, and has a certain limit with the surrounding normal bone tissues, the ASC-CM group has obviously increased new tissues, the new bone tissues can be seen, and the antler stem cell conditioned medium obviously enhances the periodontal bone regeneration.
Micro-CT scanning
Micro-CT analysis of collected rat Bone Tissue samples was performed, and the results are shown in fig. 2, wherein BV/TV (Bone Volume/Tissue Volume, Bone Volume fraction) of ASC-CM group was significantly increased (p <0.05) compared to PBS and α -MEM group; TBTH (Trabecular Thickness) of the ASC-CM group is also obviously higher than that of a normal saline group (p <0.01), which indicates that the antler stem cell secretes protein to effectively promote regeneration of periodontal bone tissue.
Example 3 the conditioned Medium of deer antler Stem cells promotes osteogenic differentiation of bone marrow mesenchymal Stem cells and inhibits osteoclast formation
1. Culture of rat bone marrow mesenchymal stem cells and rat bone marrow mononuclear cells
2 SD rats of 4 weeks old are purchased, rat bone marrow mesenchymal stem cells and rat bone marrow mononuclear cells are separated and primarily cultured and are marked as P0 generation, and the following experiment is carried out after two primary cultured cells are passed, namely P1 generation. The specific isolation and primary culture methods were performed according to the following references, respectively:
sing Ming, Liangyuling, Huang wen bin, Cheng, Jiang Sheng, He Gu Lin, Gaoyi, Panming Xin, Galectin-3 induces rat mesenchymal stem cells to differentiate into liver-like cells, Wai Chi university report, 2018, 38(9): 1076-1082.
Wangweining, Maryong, Zhengsuyang, Guoshu, Yuanhan, Sunji, a method for rapid isolation of rat bone marrow mononuclear cells and induced differentiation into osteoclasts, journal of modern medicine in China, 2019, 27(18).
2. Cell proliferation assay (CCK-8 and EDU)
The mesenchymal stem cells are inoculated on a 98-pore plate by 5000 cells/pore, the cells are treated in three groups, each group has 6 pores, the liquid is changed every day and the cells are treated, the first group is a PBS group, and the components of a culture medium of each pore are as follows: 200 u L DMEM complete medium +10 u L PBS buffer solution; the second group was the α -MEM group, with the media composition per well: 200. mu.l DMEM complete medium + 10. mu.l alpha-MEM; the third group is an ASC-CM group, and the culture medium components of each well are as follows: 200 μ l DMEM complete medium +10 μ l ASC-CM concentrate. And (3) carrying out CCK-8 detection on days 1, 3 and 5 after inoculation respectively, wherein the detection steps are as follows: preparing 10% CCK-8 incubation solution with DMEM complete culture medium, removing the culture medium from the wells to be detected, adding 100 μ l CCK-8 incubation solution into each well, 37 deg.C, and 5% CO2And (5) incubating the culture box for 2 hours, and detecting the OD value at 560nm by using an enzyme-labeling instrument.
The results are shown in fig. 3, and the OD values of the ASC-CM groups at days 1, 3 and 5 are all significantly higher than those of the other two groups, and the differences are statistically significant, and the differences between the PBS group and the α -MEM group are not significant, which indicates that the conditioned medium of the antler stem cells can significantly promote the proliferation of the bone marrow mesenchymal stem cells.
3. Osteogenic differentiation experiment
Bone marrow Mesenchymal Stem Cells (MSCs) at 1 × 104Inoculating each cell/well into a 6-well plate, culturing for 1-2 days to ensure that the cell fusion degree is 60% -70%, replacing with an osteogenesis induction culture medium, and dividing into three groups, wherein the first group is a PBS group, and the osteogenesis induction culture medium in each well comprises the following components: 2ml of DMEM complete medium + 100. mu.l of PBS buffer solution +100nmol/L dexamethasone +0.05mmol/L ascorbic acid +10mmol/L sodium beta-glycerophosphate; the second group is an alpha-MEM group, and the medium composition of the osteogenesis induction medium in each hole is as follows: 2ml of DMEM complete medium + 100. mu.l of alpha-MEM +100nmol/L dexamethasone +0.05mmol/L ascorbic acid +10mmol/L sodium beta-glycerophosphate; the third group is ASC-CM group, and the culture medium composition of osteogenesis induction culture medium in each hole is as follows: 2ml DMEM complete medium + 100. mu.l ASC-CM concentrate +100nmol/L dexamethasone +0.05mmol/L ascorbic acid +10mmol/L sodium beta-glycerophosphate. Changing the solution every 3 days, respectively performing alkaline phosphatase staining on days 3 and 7, and performing alkaline phosphatase staining on day 21Alizarin red staining was performed to evaluate osteogenic differentiation.
The results are shown in FIG. 4, which shows that the ASC-CM group with alkaline phosphatase calcium cobalt staining on day 3 has a small amount of gray staining, the staining of the other two groups is not obvious, the ASC-CM group with day 7 has a large amount of black and gray particles, and the staining of the other two groups has only a small amount of black and gray particles, and the difference is obvious; as shown in fig. 5, alizarin red staining at day 21 shows that red calcium nodule particles are present in the ASC-CM group, which are significantly greater than those in the PBS group and the α -MEM group, and the difference is statistically significant, which indicates that the conditioned medium of the deer antler stem cells can significantly promote osteogenic differentiation of the bone mesenchymal stem cells.
4. Osteoclast differentiation assay
Bone marrow mononuclear cells at 1.5X 105cells/cm2The cells were inoculated in a six-well plate until the cells grew to an appropriate density, and the osteoclast induction medium was replaced with three groups, the first group being a PBS group, and the osteoclast induction medium of each well had the following composition: 2ml DMEM complete medium + 100. mu.l PBS buffer +25ng/ml M-CSF +100ng/ml RANKL; the second group was the α -MEM group, and the osteoclast-inducing medium composition per well was: 2ml DMEM complete medium + 100. mu.l alpha-MEM +25ng/ml M-CSF +100ng/ml RANKL; the third group is an ASC-CM group, and the ingredients of an osteoclast induction culture medium in each hole are as follows: 2ml DMEM complete medium + 100. mu.l ASC-CM concentrate +25ng/ml M-CSF +100ng/ml RANKL. The solutions were changed every other day and TRAP anti-tartrate acid phosphatase staining was performed on day 6 of induction.
The results are shown in fig. 6, the PBS group and the α -MEM group showed multinucleated osteoclast formation, ASC-CM osteoclast formation was significantly less than those of the other two groups, and the difference was statistically significant, indicating that the conditioned medium of the deer antler stem cells inhibited osteoclast differentiation.
In conclusion, the antler stem cell conditioned medium can promote regeneration of periodontal bone tissues, promote proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells, and inhibit differentiation of monocytes to osteoclasts, thereby promoting bone regeneration.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (10)
1. Application of cornu Cervi Pantotrichum stem cell conditioned medium in preparing medicine for treating bone injury is provided.
2. The use of claim 1, wherein the bone injury comprises a periodontal bone injury.
3. Application of the antler stem cell conditioned medium in preparing products for promoting stem cell proliferation.
4. Application of the antler stem cell conditioned medium in preparing products for promoting osteogenic differentiation of stem cells.
5. The use of claim 3 or 4, wherein the stem cell is a bone marrow mesenchymal stem cell.
6. Application of the conditioned medium of the pilose antler stem cells in preparing products for inhibiting differentiation of monocytes into osteoclasts.
7. The use according to any one of claims 1 to 6, wherein the preparation method of the antler stem cell conditioned medium comprises: and (3) centrifugally separating the antler stem cell culture product to obtain a supernatant, and performing ultrafiltration concentration to obtain the antler stem cell conditioned medium.
8. Use according to claim 7, wherein the ultrafiltration concentration has a molecular weight cut-off of 3 kDa.
9. The use of claim 7, further comprising, after ultrafiltration concentration, the step of lyophilizing to obtain a lyophilized powder, wherein the lyophilized powder is frozen at-80 ℃ before lyophilization and the pre-cooling temperature for lyophilization is-45 ℃.
10. A product for treating periodontitis comprises a pilose antler conditioned stem cell conditioned medium.
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| CN114569634A (en) * | 2022-03-14 | 2022-06-03 | 广州医科大学附属口腔医院 | Application of antler mesenchymal stem cell conditioned medium in periodontitis drugs |
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| KR20180047952A (en) * | 2016-11-02 | 2018-05-10 | (주)프로스테믹스 | Functional composition comprising deer antlers derived stem cell culture medium |
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| CN114569634A (en) * | 2022-03-14 | 2022-06-03 | 广州医科大学附属口腔医院 | Application of antler mesenchymal stem cell conditioned medium in periodontitis drugs |
| CN114569634B (en) * | 2022-03-14 | 2024-03-12 | 广州医科大学附属口腔医院 | Application of pilose antler mesenchymal stem cell conditioned medium in periodontitis medicine |
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